The goal of this project is to combine a powerful protease profiling technology with protein engineering to develop a designer protease with novel extended substrate specificity for the treatment of cancer. Inactivation of signaling through the VEGFR-2/FIk-1 receptor has a potent anti-angiogenic effect on tumor vasculature, leading to the systematic death of the tumor. Current small molecule and antibody efforts have been unable to fully inhibit the VEGF signaling in clinical trials. Thus the inhibition of angiogenic signaling through FIk-1 represents an underdeveloped therapeutic area. Due to their catalytic nature and smaller size, engineered proteases as a therapeutic modality have a number of advantages over competing platforms including better tumor penetration, better target saturation, higher effectiveness, and potentially lower dosing. By starting with natural proteases that have high serum half-lives and reduced inhibitor binding, we will design a serine protease with an in vitro specificity that matches the FIk-1 stalk over a six amino acid region using the powerful pairing of protein engineering techniques and a proprietary protease substrate profiling technology. Positional scanning synthetic combinatorial library (PSSCL) profiling allows one to generate a complete substrate specificity profile or "fingerprint" of a protease using a single 96-well plate. PSSCL can be used to track the change in the specificity profile of variant proteases. Therefore, therapeutically relevant second generation molecules can be identified which have enhanced specificity toward target substrates and diminished specificity towards alternate substrates. Variant proteases will be extensively tested for their ability to effectively hydrolyze FIk-1, abrogate intracellular signaling, and reduce VEGF levels through binding of the soluble receptor fragment. The best candidate for clinical development will be an engineered protease that irreversibly inactivates the target receptor leading to a potent anti-angiogenic effect.